Myocardial grafts and cellular compositions

ABSTRACT

Described are preferred myocardial grafts of skeletal myoblasts or cardiomyocytes, and cellular compositions and methods useful in obtaining the grafts. The myocardial grafts are stable and can be used, for example, to deliver recombinant proteins directly to the heart.

This application is a division of application Ser. No. 08/153,664, filedNov. 16, 1993.

BACKGROUND OF THE INVENTION

The present invention resides generally in the field of cardiology, andmore particularly relates to stable myocardial grafts and methods andcellular compositions useful for achieving such grafts.

As further background, organ transplantation has been widely used toreplace diseased, nonfunctional tissue. More recently, cellulartransplantation to augment deficiencies in host tissue function hasemerged as a potential therapeutic paradigm. One example of thisapproach is the well publicized use of fetal tissue in individuals withParkinsonism (reviewed in (1), see reference list, infra.), wheredopamine secretion from transplanted cells alleviates the deficiency inpatients. In other studies, transplanted myoblasts from uneffectedsiblings fused with endogenous myotubes in Duchenne's patients;importantly the grafted myotubes expressed wild-type dystrophin (2).

Despite their relevance in other areas, these earlier studies do notdescribe any cellular transplantation technology which can besuccesfully aplied to the heart, where the ability to replace damagedmyocardium would have obvious clinical relevance. Additionally, the useofintra-cardiac grafts to target the long-term expression of angiogenicfactors and ionotropic peptides would be of therapeutic value forindividuals with myocardial ischemia or congestive heart failure,respectively.

In light of this background there is a need for the development ofcellular transplantation technology in the heart. Desirably, suchtechnology would not only provide stable grafts in the heart but alsoenable the delivery of useful recombinant proteins or other moleculesdirectly to the heart. The present invention addresses these needs.

SUMMARY OF THE INVENTION

The applicant has established cellular grafts in the myocardium whichare viable long-term. Cardiomyocytes and skeletal myoblasts have beengrafted directly into the myocardium of syngeneic animals. Viable graftswere detected at least one-half year post-implantation (the latest timepoint assayed). The presence of the grafts was not accompanied by overtcardiac arrhythmia, and the majority of the grafts were juxtaposeddirectly to the host myocardium and not encapsulated. It has thus beendiscovered that the myocardium can serve as a stable platform forcellular transplants. These transplants can be used for the localdelivery of recombinant molecules to the heart and/or for replacingdiseased tissue to supplement myocardial function.

Accordingly, one preferred embodiment of the invention provides amyocardial graft in an animal which includes a stable graft of skeletalmyoblasts or cardiomyocytes incorporated in myocardial tissue of theanimal.

Another preferred embodiment of the invention provides a method forforming a stable myocardial graft in an animal. The inventive methodincludes the step of introducing skeletal myoblasts or cardiomyocytes inmyocardial tissue of the animal so as to form a stable myocardial graft.The cells can be conveniently introduced, for example, by injection.

Another preferred embodiment of the invention provides a method fordelivering a recombinant molecule to myocardial tissue of an animal.This method includes the step of establishing a stable graft of skeletalmyoblasts or cardiomyocytes incorporated in myocardial tissue of theanimal, wherein the myoblasts or cardiomyocytes deliver the recombinantmolecule to the myocardial tissue. In this embodiment the myoblasts orcardiomyocytes will carry transgenes encoding the recombinant molecule.

Another preferred embodiment of the invention provides a cellularcomposition comprising a substantially homogeneous population ofnon-immortalized cardiomyocytes. This and other cell populations can beobtained utilizing a preferred inventive method that includes (i)transfecting embryonic stem cells to introduce a marker gene enablingselection of one cell lineage from other cell lineages resulting fromdifferentiation of the stem cells, (iii) causing the stem cells todifferentiate, and (iv) selecting said one cell lineage based on themarker gene. The cells used in and resulting from such methods also forma part of the present invention.

Still another preferred embodiment of the invention provides a non-humananimal having a stable graft of skeletal myoblasts or cardiomyocytesincorporated in myocardial tissue of the animal.

The invention thus provides myocardial grafts, methods and cellularcompositions useful for forming myocardial grafts, and animals whichhave the myocardial grafts. The grafts will find use both as a vehiclefor delivering therapeutic substances such as recombinant proteins andother molecules, and as a means for replacing diseased tissue tosupplement myocardial function. Cellular compositions of the inventioncan be used directly to prepare grafts, and will also be useful inscreening drug substance effects on cardiomyocytes and for expressingand obtaining recombinant proteins. Grafted animals can be used, forexample, to screen the effects of recombinant molecules on the heart.

These and other objects and advantages of the invention will be beapparent from the following description.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a schematic diagram illustrating DNA used to generate MHC-nLACtransgenic mice in Example 3, infra.

DESCRIPTION OF THE PREFERRED EMBODIMENT

For the purpose of promoting an understanding of the principles of theinvention, reference will now be made to certain embodiments thereof andspecific language will be used to describe the same. It willnevertheless be understood that no limitation of the scope of theinvention is thereby intended, such alterations, further modificationsand applications of the principles of the invention as illustratedherein being contemplated as would normally occur to one skilled in theart to which the invention relates.

As indicated above, the present invention provides stable myocardialgrafts of skeletal myoblasts and/or cardiomyocytes. In this regard, asused herein the term "stable myocardial graft" is intended to mean amyocardial graft whose cells are viable for a period of at least about 2weeks. Surprisingly, such stable grafts have been readily achieved inaccordance with the invention, with preferred grafts having cells viablefor six months or more. Myocardial grafts of the invention can thusprovide for long-term delivery of recombinant proteins or othermolecules to the heart and/or long-term supplementation of myocardialtissue.

The skeletal myoblasts and cardiomyocytes used in the invention can beobtained or isolated from any suitable source. Skeletal myoblasts,including for example C2C12 skeletal myoblasts, are available frompublic depositories such as the American Type Culture Collection (ATCC)(Rockville, Md.). Skeletal myoblasts can also be isolated from skeletalmuscle using techniques well known to the art and literature.Cardiomyocytes useful for the invention can be obtained using techniquesdescribed in the literature (3) or using methods described moreparticularly in the Examples below. Briefly, one such method involvesdigestion of heart tissues to obtain cardiomyoctes.

Another method involves using an appropriate marker to select specificcell lineages, such as cardiomyocytes, from other cell lineagesresulting from the differentiation of embryonic stem cells (totipotentcell lines derived from the inner cell mass of blastocysts as describedin (22)). The preferred method involves a positive selection scheme.Thus, a marker gene, such as a gene conferring antibiotic resistance(e.g. neomycin or hygromycin), is introduced into the stem cells underappropriate control such that expression of the gene occurs only in thedesired cell lineage. For example, the marker gene can be under thecontrol of a promoter which is active only in the desired cell lineage.Upon differentiation of the stem cells, the desired lineage is thenselected based upon the marker, e.g. by contacting the mixeddifferentiated cells with the appropriate antibiotic to which thedesired lineage has been conferred resistance. Cell lines other than thedesired line will thus be killed, and a substantially pure, homogeneouspopulation of the desired line can be recovered. In more preferredmethods, two markers are introduced into the parent stem cells, oneallowing selection of transfected stem cells from non-transfected cells,and one allowing selection Of the desired cell lineage from otherlineages. A double positive selection scheme can thus be used where eachselectable marker confers antibiotic resistance. Using this selectionmethodology, populations comprised about 90% and even about 95-100% ofthe desired cell lineage can be obtained, as demonstrated in theExamples below.

To obtain grafts of the invention, the skeletal myoblasts orcardiomyocytes will be introduced into the myocardial tissue of a livinganimal such as a mammal. The cells can be introduced in any suitablemanner, but it is preferred that the mode of introduction be asnon-invasive as possible. Thus, delivery of the cells by injection,catheterization or similar means will be more desired.

The resulting graft-bearing animals have exhibited normal sinus rhythms,indicating that the graft per se, as well as the graft-host myocardiumborder zone, does not induce arrhythmias. This is in stark contrast tothe remodeling that frequently occurs following infarcts in humans; theborder zone of the infarct may give rise to circus loops which result inclinically significant arrhythmias (4, 5).

Grafts of the invention can be proliferative or non-proliferative. Forexample, the AT-1 grafts established in the specific Examples below areproliferative. On the other hand, the skeletal myoblast-derived graftsformed in the Examples are non-proliferative, with the absence oftritiated thymidine uptake demonstrating that the formation of stableintra-cardiac grafts was not dependent upon sustained cellproliferation.

Preferred grafts will be characterized by the presence of directintracellular coupling and the formation of gap junctions between hostand grafted cells. Moreover, such grafts will not cause immune responsein the host, and will exhibit terminal differentiation of grafted cellsand a non-tumorogenic nature.

Grafts of the invention are useful inter alia to deliver therapeuticproteins and the like via secretion from grafted cells, and to replacediseased or damaged tissue to supplement myocardial function. Asexamples of therapeutic protein deliveries, grafts may expressangiogenic factors (as exemplified by basic and acidic Fibroblast GrowthFactor; Transforming Growth Factor--Beta, Vascular Endothelial GrowthFactor and Hepatocyte Growth Factor) to induce neovascularization.Similarly, grafts expressing neurotrophic agents near an infarctedregion may be used to ameliorate the arrhythmogenesis associated withthe border zone. These and many other candidate substances for targeteddelivery to the heart will be apparent to those skilled in the area.

To promote a further understanding of the invention and its principlesand advantages, the following specific Examples are provided. It will beunderstood that these Examples are illustrative, and not limiting, innature.

EXAMPLE 1 Generation of Stable AT-1 Cardiomyocyte Grafts

A. METHODS

AT-1 Cell Culture and Myocardial Grafting Protocol AT-1 cardiomyocyteswere isolated from subcutaneous tumors by sequential collagenasedigestion and cultured in PC-1 medium (Ventrex, Coon Rapids Minn.)containing 10% fetal calf serum as previously described in (6). Cellswere labeled with 10 μM8-chloromethyl-4,4-difluoro-1,3,5,7,-tetramethyl-4-bora-3a,4a-diazaindecene(BODIPY, Molecular Probes, Eugene Oreg.) for 30 min at 37° C. tofacilitate localization of the injection site. Immediately beforeinjection, cells were harvested with trypsin and collagenase, washedthree times with serum-free PC-1 medium and directly injected into theventricular myocardium of syngeneic B6D2/F1 mice (Jackson Laboratories,Bar Harbor Mass.) under open heart surgery as described in (7). Cells(4-10×10⁴) were injected in a volume of 2-3 μl using a plastic syringefitted with a 30 gauge needle.

Histology. Hearts were removed following cervical dislocation andcryoprotected in 30% sucrose, embedded and sectioned at 10 μm with acryomicrotome as described (8). For hematoxylin and eosin (H and E)staining, sections were post fixed in acetone:methanol (1:1) and stainedaccording to manufacturer's specifications (Sigma Diagnostics, St. LouisMo.). For immuno-histology, unfixed sections were reacted withpolyclonal rabbit anti-T-Ag antibodies (either 161-T, see (3) or 162-T)followed by horseradish peroxidase-conjugated goat anti-rabbit antisera(Boehringer Mannheim, Indianapolis Ind.), and visualized bydiaminobenzidine reaction with nickel enhancement as described in (9).Monoclonal antibodies against the common leukocyte antigen (CD45;antibody M1/9.3 HL, Boehringer Mannheim) and against the macrophageMac-1 antigen (CD11b; antibody M1/70 HL, Boehringer Mannheim) were usedto monitor intra-cardiac graft rejection. The Mac-1 antibody has 75-90%cross reactivity with lymphocytes. After treatment with primaryantibody, sections were incubated with horseradish peroxidase-conjugatedrabbit anti-rat antisera (Boehringer Mannheim), and visualized bydiaminobenzidine reaction with nickel enhancement. For ³ H!-thymidineincorporation, mice were given a single bolus injection of isotope (400μCi at 28 Ci/mM, Amersham, Arlington Heights Ill.) and eighteen hourslater sacrificed by cervical dislocation. The heart was removed,cryoprotected in 30% sucrose, embedded and sectioned with acryomicrotome. Sections were post-fixed in methanol:acetone (1:1),stained with H and E, and a thin layer of photographic emulsion (IlfordL.4, Polysciences, Warrington Pa.) diluted 1:1 with distilled water wasapplied. Sections were exposed for 5-7 days at 4° C., and developed inKodak D-19 at 20° C. for 4 minutes, washed with distilled water for 1minute, fixed in 30% sodium thiosulfate for 10 minutes, and washed indistilled water.

Electron Microscopy (EM). Tissue blocks were fixed in 2% glutaraldehydein 0.1M cacodylate buffer (pH 7.4) and post-fixed in 2% osmium tetroxide(Stevens Metallurgical Corp., New York N.Y.). All other EM chemicalswere obtained from Ladd Research Industries, Inc. (Burlington Vt.).Tissue was stained en bloc with 2% uranyl acetate in pH 5.2 maleatebuffer (0.05M), dehydrated, and embedded in Ladd LX-112. Grafts werelocated using 1 μm sections stained with toluidine blue. After trimming,the block was thin sectioned, and stained with uranyl acetate and leadcitrate. Specimens were viewed on a Phillips 400 transmission electronmicroscope.

Electrocardiogram (ECG) Analyses. For surface ECG records, mice wereanesthetized (2.5% Avertin,0.015 ml/g body weight, IP, Fluka Chemicals,Lake Ronkomkoma N.Y.), surface electrodes were placed in the standardlead 1 position, and ECGs were recorded with a Narco Biosystems (HoustonTex.) high gain amplifier coupled to an A/D converter (CoulbournInstruments, Lehigh Valley Pa.).

Plasma Enzyme Assay (PEA). For lactate dehydrogenase (LDH) isoformassay, plasma was isolated by retro-orbital sinus bleeds underanesthesia (2.5% Avertin,0.015 ml/g body weight, intraperitoneally(IP)). Plasma was fractionated on 1% agarose gels (CK Isoenzymeelectrophoresis system, CIBA-Corning Diagnostics, Corning N.Y.) and theLDH isoforms visualized by a TNBT-Formazan histochemical assay (LDHAssay Kit, Sigma Diagnostics, St. Louis Mo.).

B. RESULTS

In these studies, AT-1 cardiomyocytes (derived from transgenic animalsthat expressed the T-Ag oncoprotein in the heart) were injected directlyinto the myocardium of syngeneic mice and the viability of the graftedmaterial was assessed. To facilitate localization of the injection sitein preliminary experiments, AT-1 cardiocytes were incubated briefly withBODIPY prior to grafting. BODIPY is a nontoxic glutathione reactive dyewhich permits fluorescent tracking of living cells. The graft site waseasily visualized by fluorescence microscopy using a FITC cube.Subsequent experiments did not utilize BODIPY.

Fifty percent (14/28) of the animals receiving AT-1 cardiomyocyteinjections developed intra-cardiac grafts. In most instances, the graftswere neither encapsulated nor surrounded by scarred myocardium. At thelevel of light microscopy, grafted AT-1 cardiomyocytes were observeddirectly juxtaposed with host cardiomyocytes. The identity of the AT-1cardiomyocytes was confirmed by immuno-peroxidase assay using ananti-T-Ag antibody primary antibody (162-T) followed by a horseradishperoxidase conjugated secondary antibody. Specificity of the anti-T-Agantibody has been established previously (10, 11). Black precipitate towas observed over cardiomyocyte nuclei in the graft but not in the hostmyocardium, confirming that the graft was comprised of AT-1cardiomyocytes. Similar results were obtained with other anti-T-Agantibodies, and no signal was observed in the absence of primaryantibody.

Viable AT-1 cardiomyocytes were observed at least as long as four monthspost-implantation. During this period, some degree of graftproliferation occurred; ³ H!-thymidine incorporation analyses detectedDNA synthesis in the grafted cells. Ten percent of the AT-1cardiomyocyte nuclei were synthesizing DNA as evidenced by isotopeincorporation into the nucleus. However, the rate was appreciably lessthan that observed for cultured AT-1 cardiomyocytes, where 50% of thecells synthesized DNA following a similar ³ H!-thymidine pulse. Inseveral instances, the grafted AT-1 cardiomyocytes were localized withinthe subpericardial space.

Immunohistologic experiments were employed to determine if the intra-cardiac grafts were subject to chronic rejection. Grafts older than onemonth failed to react with antibodies specific for mouse leukocytes;signals observed in blood vessels located on the same section provided apositive control for the experiment. Similarly, an antibody whichdetects mouse macrophages and lymphocytes did not react with theintra-cardiac graft; once again positive signal was observed in a bloodvessel located on the same section. Collectively, these results indicatethe absence of chronic graft rejection by the syngeneic hosts. Thisresult is supported by the observation that cyclosporine treatment (50mg/kg body weight, administered intraperitoneally daily) did notinfluence significantly the frequency of intra-cardiac grafting (50%success rate, n=6). Sex of the host animal also did not appear toinfluence significantly the rate of graft formation (46% success rate inmales, n=13; 53% success rate in females, n=15). The frequency ofgrafting was similar in animals examined at early time points (1-40 dayspost-grafting, 47%, n=15) as compared to those examined at later timepoints (40-120 days post-grafting, 54%, n=13). Finally, similarfrequencies of intra-cardiac grafting were observed when cells weredelivered to either the left ventricular free wall or the apex of theheart.

Electron microscopic analysis of the AT-1 cardiomyocyte grafts confirmedthe absence of encapsulation. High power views revealed well-developedjunctional complexes between adjacent cells within the graft. Graftcardiomyocytes contained numerous polyribosomes and the dedifferentiatedmyofibrillar ultrastructure typical of AT-1 tumors in vivo (6).Electron-dense secretory granules were also observed in the AT-1cardiomyocyte grafts, as would be expected for myocytes of atrialorigin. Host cardiomyocytes bordering the grafts had normalultrastructure with well-formed sarcomeres. Although only a thinbasement membrane separated AT-1 and host cardiomyocytes, no junctionalconnections between these two cell types were observed.

Surface electrocardiograms were performed to determine if the presenceof AT-1 cardiomyocyte grafts influenced the autonomic rhythm. Noappreciable differences were observed between records from sham animalsand those which harbored grafts. In each case, the experimental animalsexhibited normal sinus rhythm, with an anesthetized heart rate ofapproximately 400 beats per minute. Normal P-QRS coupling wasmaintained, indicating that the grafted AT-1 cardiomyocytes did not actas an ectopic pacemaker. This latter result is important in light of theobservation that AT-1 cardiomyocytes exhibit spontaneous electricalactivity both in vivo (12) and in culture (3). The absence of overtarrhythmia also indicated that graft-induced myocardial remodeling wasnot associated with the generation of significant circus rhythms.

In addition to surface ECG, plasma LDH levels were assessed in micecarrying AT-1 cardiomyocyte grafts. The presence of the cardiac LDHisoform in the circulation is a well established hallmark of myocardialinfarction. No cardiac LDH (isoform-1) was apparent in mouse plasmaprior to grafting. After the introduction of AT-1 cardiomyocytes, therewas a transient appearance of the cardiac isoform in the Plasma, whichmost likely reflected damage to the host myocardium as well as damagedAT-1 cardiomyocytes. A transient increase in plasma skeletal LDH isoformwas also observed following grafting surgery, presumably reflectingdamage caused by the trans-thoracic incision. The plasma LDH profilesreturned to normal by 7 days post-implantation. Thereafter, the plasmaLDH profiles remained normal despite the presence of grafts.

EXAMPLE 2 Generation of Stable C2C12 Myoblast Grafts

A. METHODS

C2C12 Cell Culture and Myocardial Grafting Protocol. C2C12 myoblastswere obtained from ATCC. Cells were maintained in the undifferentiatedstate by culturing at low density in high glucose Dulbecco's ModifiedEagle Media (DMEM) supplemented with 20% fetal bovine serum, 1% chickenembryo extract, 100 units/ml penicillin and 100 μg/ml streptomycin. Forsome studies, myogenic differentiation was induced by culturing in DMEMsupplemented with 2% horse serum and antibiotics. Immediately beforeinjection, myoblasts were harvested with trypsin, washed three timeswith serum free DMEM and directly injected into the ventricularmyocardium of adult syngeneic C3Heb/FeJ mice (Jackson Laboratories)under open heart surgery as described in (7). Cells (4-10×10⁴) wereinjected in a volume of 2-3 μl using a plastic syringe fitted with a 30gauge needle.

Histology. Hearts were removed, cryoprotected, embedded and sectioned asin Example 1. H and E staining and ³ H!-thymidine incorporation assayswere also conducted as in Example 1. For immunohistology, methanol fixedsections (-20° C., 10 min.) were reacted with the monoclonalanti-skeletal myosin heavy chain antibody (MY-32, Sigma Chemical Corp.)followed by rhodamine-conjugated sheep anti-mouse IgG F(ab')₂ fragment(Boehringer Mannheim), and visualized by epifluorescence.

Electron Microscopy. EM was performed as in Example 1.

Electrocardiogram Analyses. ECG anlyses were performed as in Example 1.

Plasma Enzyme Assay. PEA was performed as in Example 1.

B. RESULTS

Several myoblast cell lines are known which, as exemplified by C2C12cells, have the capacity to differentiate into myotubes in culture (13).C2C12 myoblasts were derived from cultured explants of injured thighmuscle of C3H mice. When maintained in serum-rich media, the myoblastsproliferate rapidly and retain an undifferentiated phenotype. However,when cultured in serum-poor media myogenic differentiation is induced.The C2C12 cells withdraw from the cell cycle and fuse, thereby formingmultinucleated myotubes. Myogenic differentiation is also induced, asevidenced by the appearance of numerous muscle-specific gene products.Thus, in this model proliferation and myogenic differentiation aremutually exclusive (14). Myoblast differentiation in vitro is thought tomimic satellite cell mediated myofiber regeneration in vivo.

Myoblasts were injected directly into the myocardium of syngeneicC3Heb/FeJ mice and the viability of the grafted material was assessed.One hundred percent (13/13) of the mice receiving indra-cardiac implantsof C2C12 myoblasts developed grafts in the heart. Viable grafts wereobserved as long as six months post-implantation (this was the last timepoint assayed). In all instances, the grafted material was notencapsulated. The differentiated status of the grafted C2C12 cells wasdetermined by immunohistological assay with an anti-myosin heavy chainantibody (MY-32). This antibody does not react with myoblasts nor withcardiac myosin heavy chain. Although differentiated C2C12 cells wereobserved in every heart receiving myoblast injections, the graftingefficiency of individual cells was not determined. As an additionalcontrol, hearts bearing AT-1 intra-cardiac grafts (see Example 1) wereexamined with the MY-32 antibody. No staining was observed, therebyruling out the possibility that the signal seen in the C2C12 grafts wasdue to skeletal myosin heavy chain induction in host cardiomyocytes.

Example 1 above demonstrates that AT-1 cardiomyocytes form stable graftsin syngeneic myocardium. However, the observation that these cellsretained the capacity for proliferation in vivo raised the possibilitythat sustained cell division might be required for successfulintra-cardiac grafting. The proliferative status of the C2C12 grafts wastherefore examined. Virtually no DNA synthesis (as assessed by tritiatedthymidine incorporation) was observed, indicating that the majority ofthe grafted C2C12 cells had indeed withdrawn from the cell cycle.Examination of serial sections indicated that less than 0.1% of thecells in or near the grafts were synthesizing DNA. This result mostlikely reflects fibroblast proliferation during the remodeling process.As with the AT-1 grafts, immunohistological analyses of C2C12 graftsfailed to detect macrophage, inflammatory leukocyte or lymphocyteinfiltration at two months post-implantation, indicating the absence ofchronic graft rejection by the syngeneic hosts.

At the level of light microscopy, the C2C12 intra-cardiac graftsexhibited cellular heterogeneity with both H and E and MY-32immunofluorescence staining. Electron microscopic analyses were employedin an effort to further characterize the cellular make-up of the C2C12grafts. Toluidine-stained 1 μm sections were surveyed at 100 μmintervals to locate graft sites for EM analysis. Once localized, thinsections were prepared from the block. Cells with morphology typical ofskeletal myocytes were observed throughout the graft. Abundantmitochondria localized between well developed sarcomeres were readilydetected. Prominent Z bands and thick and thin filaments were observed.Occasionally, expanded t-tubules and ruffled cell membranes weredetected in the grafted myocytes. In addition to well developedmyocytes, a second less differentiated cell type was observed in C2C12grafts. Most notably, these cells exhibited a large nucleus to cytoplasmratio, with a prominent band of heterochromatin at the nuclearperiphery. Moderate amounts of centrally located heterochromatin werealso detected. Limited rough endoplasmic reticulum and few mitochondriawere observed in these cells. Similar ultrastructural characteristicshave been ascribed to satellite cells in vivo and in culture (15, 16).

Two studies were initiated to assess any deleterious effects of C2C12intra-cardiac grafts on host heart function. In the first study, surfaceelectrocardiograms failed to detect any appreciable differences betweenrecords from control and experimental mice. All animals examined hadnormal P-QRS coupling, and exhibited normal sinus rhythm with ananesthetized heart rate of approximately 400 beats per minute. Thesedata indicate that the intra-cardiac myoblast grafts did not induceovert cardiac arrhythmias. In the second study, plasma LDH levels weremonitored in graft-bearing animals. The presence of the cardiac LDHisoform in the circulation is a well established hallmark of myocardialinfarction. The cardiac-specific LDH isoforms (isoforms 1, 2, and 3)were not observed in plasma prior to grafting. Immediately aftergrafting, an increase in the cardiac isoforms was observed in plasma,which most likely reflected damage to the host myocardium. A transientincrease in the plasma skeletal LDH isoform (isoform 5) was alsoobserved, presumably reflecting damage caused by the trans-thoracicincision. Plasma LDH profiles returned to normal by 7 dayspost-implantation.

EXAMPLE 3 Generation of Stable Fetal Cardiomyocyte Grafts

A. METHODS

Cardiomyocyte Cell Culture and Myocardial Grafting Protocol. Transgenicmice were generated which carry a fusion gene comprised of the α-cardiacmyosin heavy chain (MHC) promoter and a modified β galactosidase (nLAC)reporter. To generate the MHC-nLAC transgenic mice, MHC-nLAC insert DNA(see FIG. 1) was purified by absorption onto glass beads, dissolved at aconcentration of 5 μg/ml, and microinjected into the nuclei of one cellinbred C3H3B/FeJ embryos according to established protocols (17).Polymerase Chain Reaction (PCR) analysis was employed to identifyfounder animals and to monitor transgene segregation. The sense strandprimer 5'-GGTGGGGGCTCTTCACCCCCAGACCTCTCC-3' (SEQ. I.D. NO. 1) waslocalized to the MHC promoter and the antisense strand primer5'-GCCAGGGTTTTCCCAGTCACGACGTTGT-3'(SEQ. I.D. NO. 2) was localized to thenLAC reporter. PCR analyses were as described in (18). The MHC promoterconsisted of 4.5 kb of 5' flanking sequence and 1 kb of the geneencompassing exons 1 through 3 up to but not including the initiationcodon. The nLAC reporter was modified so as to carry both a eukaryotictranslation initiation site and the SV40 nuclear localization signal(19). The mP1 sequences carried an intron, as well as transcriptionaltermination and polyadenylation signals from the mouse protamine 1 gene.

For preparations for examination of β galactosidase (βGAL) activity andDAPI epifluorescence, transgenic animals were heparinized (10,000 U/kgIP) prior to sacrifice by cervical dislocation. Hearts were placed in abeaker of gassed (95% O₂, 5% CO₂) KHB buffer (105 mM NaCl, 20 mM NaHCO₃,3.8 mM KCl, 1 mM KH₂ PO₄, 1.2 mM MgSO₄, 0.01 mM CaCl₂, 1 mM mannitol, 10mM taurine, 10 mM dextrose, 5 mM Na-pyruvate). Hearts were then hung bythe aorta and perfused with gassed KHB (0.5 ml/min at 37° C.) containing2.5 mM EGTA for five minutes, followed by 0.17% collagenase (Type I,Worthington Biochemical, Freehold N.J.) in KHB. Hearts were perfuseduntil flaccid and the ventricles were minced with scissors and isolatedcells obtained by triturating with a Pasteur pipette. After at least onehour of formalin fixation, suspensions were filtered and smeared ontopositively charged slides (Superfrost Plus, Fisher, Pittsburgh Pa.), andallowed to dry.

For isolation of single cells for injection, females with 15 day embryos(onset of pregnancy determined by vaginal plugs) were sacrificed bycervical dislocation. Embryos were removed, decapitated, and hearts wereharvested under PBS, and ventricles and atria were separated. Transgenicventricles (identified by cardiac βGAL activity) were digested in 0.1%collagenase (Worthington) in DPBS (Dulbecco's Phosphate Buffered Saline,Sigma) for 45 minutes, and were triturated with a Pasteur pipette inPC-1 medium (Ventrex, Coons Rapids Minn.) with 10% FBS, resulting in asuspension of single cells.

Immediately after isolation, embryonic cardiomyocytes were washed threetimes with DPBS and directly injected into the ventricular myocardium ofsyngeneic mice (Jackson Laboratories) under open heart surgery as inExample 1. 1-10×10⁴ cells were injected in a volume of 2-3 μl using aplastic syringe fitted with a 30 gauge needle.

Histology. For H and E, X-GAL, immunohistology and thymidine analyses,hearts were removed following cervical dislocation and cryoprotected in30% sucrose, embedded and sectioned at 10 μm with a cryomicrotome as inExample 1. H and E staining, monitoring for intra-cardiac graftrejection, and assay for ³ H!-thymidine incorporation were alsoconducted as in Example 1. To assay βGAL activity, sections werehydrated in PBS, post-fixed in acetone:methanol (1:1) and then overlaidwith mixture containing 1 mg/ml X-GAL(5-bromo-4-chloro-3-indolyl-β-D-galactoside), 5 mM potassiumferricyanide, 5 mM potassium ferrocyanide and 2 mM magnesium chloride inPBS. Positive staining is indicated by the appearance of a bluechromophore. After treatment with primary antibody, signal wasvisualized by an avidin-biotin (ABC) kit (Vector Labs, BurlingameCalif.). The heart was processed as described above, and sections werepost-fixed in methanol:acetone (1:1), stained with H and E, and coatedwith a thin layer of photographic emulsion (Ilford L.4, Polysciences)diluted 1:1 with distilled water. Sections were exposed, developed,washed, fixed and washed as in Example 1. X-GAL staining of single cellpreparations was as described above. For visualization of nuclei insingle cell preparations, slides were stained with DAPI in PBS (0.28 μM,three min. at room temperature, Boehringer Mannheim), washed three timesin PBS, and wet-mounted in 2% propyl gallate dissolved in glycerol. Toobtain coronal heart sections, mice were sacrificed by cervicaldislocation, hearts were harvested and perfused on a Langendorffapparatus with 2% glutaraldehyde in 0.1M cacodylate buffer (pH 7.4).After immersion fixation overnight in the same buffer, 200 μm coronalsections were made with a vibratome (Campden, London, United Kingdom).To localize the graft, sections were pooled and stained for βGALactivity with X-GAL as described above.

Electron Microscopy. MHC-nLAC embryonic grafts were localized in coronalheart sections as described above. After trimming, the tissue was post-fixed in 2% osmium tetroxide (Stevens Metallurgical Corp., New YorkN.Y.). Tissue was then dehydrated and embedded in Ladd LX-112 (LaddResearch Industries). Grafted areas were further trimmed, thinsectioned, and stained with uranyl acetate and lead citrate. Specimenswere viewed on a Phillips 400 transmission electron microscope as inExample 1.

Electrocardiogram Analyses. ECG analyses were conducted as in Example 1.

B. RESULTS

Transgenic mice generated as above carried a fusion gene comprised ofthe MHC promoter and a nLAC reporter. nLAC carries the SV40 nucleartransport signal, which results in the accumulation of β galactosidaseactivity in the nucleus of targeted cells. Four transgenic lineages wereproduced, and two (designated MHC-nLAC-2 and MHC-nLAC-4) were selectedfor further analyses. To ensure that the MHC-nLAC transgene wouldprovide a suitable cell lineage marker, β galactosidase (βGAL) activitywas assessed in transgenic cardiomyocytes. Single cell preparationsgenerated by retrograde collagenase perfusion were examinedsimultaneously for βGAL activity and DAPI epifluorescence. 99.0±0.45%(n=400) of the transgenic cardiomyocyte nuclei expressed βGAL, whereasno βGAL activity was detected in noncardiomyocytes. In addition, nonuclear βGAL activity was detected in nontransgenic controlcardiomyocytes.

Single cell suspensions were prepared by collagenase digestion of heartsharvested from embryonic day 15 transgenic mice. Greater than 95% of thecardiomyocytes isolated by this technique were viable as evidenced bydye exclusion assay. Cardiomyocytes were delivered to left ventricularfree wall of syngeneic nontransgenic animals. Grafted cardiomyocyteswere readily and unambiguously identified by virtue of the nuclear βGALactivity encoded by the MHC-nLAC transgene. Grafted cardiomyocytes werefrequently observed at sites distal to the point of delivery; itpresently is not clear if this distribution of grafted cells reflectscardiomyocyte migration or passive diffusion along dissection planesproduced by the injection process. Approximately 50% (7/13) of theanimals receiving intra-cardiac injections of embryonic cardiomyocytesdeveloped grafts. This frequency of successful graft formation is likelyto increase as cell preparation and implantation protocols areoptimized.

Light microscopic analyses of H and E stained sections processed forβGAL activity indicated that grafted cardiomyocytes (blue nuclei) werejuxtaposed directly with host cardiomyocytes (purple nuclei). AdditionalH and E analyses failed to detect significant graft encapsulation. Theobserved proximity of graft and host cardiomyocytes and absence ofencapsulation are prerequisites for successful coupling between the twocell types.

Consecutive sections of a 19 day old intra-cardiac graft were processedfor H and E, βGAL activity, and macrophage and leukocyteimmunoreactivity. No evidence for graft rejection was observed, despitethe fact that the animals were not immune suppressed. As a positivecontrol for the immunohistology, grafts of incompatible MHC haplotypewere produced; graft rejection was clearly evident in these hearts.Tritiated thymidine uptake analyses indicated that only 0.6% (n=156) ofthe βGAL-positive nuclei were synthesizing DNA, althoughnoncardiomyocyte DNA synthesis was apparent. Since the embryonic day 15donor cells were still mitotically active when grafted (labeling indexof ca. 29%), the exceedingly low level of DNA synthesis observed in βGALpositive cells at 19 days post-grafting suggested that the MHC-nLACembryonic cardiomyocytes had undergone terminal differentiation.

The juxtaposition of graft and host cardiomyocytes observed by lightmicroscopic analyses prompted a determination whether directintercellular coupling could be detected between the two cell types. TheX-GAL reaction product is an electron-dense precipitate which can bedetected by transmission electron microscopy (TEM, see 19). Vibratomesections from glutaraldehyde perfusion-fixed hearts were stained forβGAL activity, and grafted regions thus identified were trimmed andembedded for TEM. βGAL positive nuclei were readily observed by lightmicroscopic analysis of 1 μm sections. The X-GAL reaction product had aperinuclear appearance due to a slight degree of nuclear leaching whichoccurred during the embedding process. The same groups of cardiomyocyteswere identified by TEM analysis of a consecutive thin section. Hostcardiomyocytes, which were not readily identified in the lightmicrographs due to the absence of perinuclear βGAL activity, wereobserved by electron microscopy to be juxtaposed with the grafted cells.Numerous junctional complexes were present between the host and graftcardiomyocytes, indicating a high degree of intercellular coupling. Manyexamples of intercellular coupling between host and graft cardiomyocyteswere observed throughout the grafted regions. Importantly, intercellularconnections could be traced from βGAL positive cardiomyocytes throughnumerous host cells, thus demonstrating that grafted cardiomyocytescould be participating in a functional syncytium.

In addition to documenting the presence of abundant intercellularcoupling between grafted and host cardiomyocytes, the TEM analysesrevealed that the grafted cardiomyocytes were highly differentiated.Normal characteristics of adult cardiomyocytes were observed includingmyofibrillae forming complete sarcomeres, numerous junctional complexesbetween cells and abundant mitochondria. Indeed, aside from the presenceof the X-GAL reaction product, grafted cardiomyocytes wereindistinguishable from host cells. Further, binucleated, βGAL positivecells could be detected in the intra-cardiac grafts. Becausebinucleation is a characteristic of adult rodent cardiomyocytes, thisobservation further supports that the grafted cardiomyocytes haveundergone terminal differentiation.

Surface ECG recordings were employed to determine if the presence ofcoupled embryonic cardiomyocyte grafts negatively influenced host heartautomaticity. ECG traces from graft-bearing animals wereindistinguishable from sham operated controls, and exhibited P and QRScomplexes typical for mice. There was no evidence for cardiac arrhythmiain graft-bearing animals, despite the presence of a high degree ofintercellular coupling between grafted and host cardiomyocytes.

EXAMPLE 4 Preparation of Substantially Pure Cardiomyocyte Culture

Embryonic stem cells were genetically modified in a manner enabling theproduction of a substantially homogeneous population of non-immortalizedcardiomyocytes. The parental ES cell line (D3) was cotransfected with apGK-HYG (hygromycin) plasmid and a plasmid containing a MHC-neo^(r)gene. The pGK-HYG plasmid provides selection for transfected ES cells,while the mMHC-neo^(r) gene facilitates a second round of selection ondifferentiated cells: incubation ill the presence of G418 eliminatesnon-cardiomyocytes (that is, cells in which the MHC promoter is notactive).

Stably tranfected ES cells were selected by growth in the presence ofhygromycin. The plasmids were linearized and introduced into the stemcells via electroporation at 1180 μFarad, 220 volts. The transfectedcells were maintained in DMEM supplemented with 10% preselected FBS, 0.1mM β-mercaptoethanol, nonessential amino acids, PenStrep and LIF, andtransformants selected by the addition of hygromycin into the medium.Co-transfectants were then identified by PCR analysis specific for bothtransgenes. The transfections produced a cell line, designated 9A, whichcarries both transgenes.

Cardiogenesis was induced in 9A ES cells by plating 2×10⁶ cells ontouncoated 100 mm bacterial petri dishes in the absence of LIF. After 8days in culture, numerous patches of cells exhibiting spontaneouscontractile activity (i.e. cardiomyocytes) were observed. At this point,G418 was added to the media, and the cells incubated for an additional 9days. During this treatment it was apparent that many of thenon-cardiomyocytes were being killed by the G418. Importantly, the G418had no discernible effects on the cardiomyocytes, which retained theirspontaneous beating activity throughout the course of the experiment.

After a total of 9 days of G418 selection, the surviving cells weredissociated with collagenase and trypsin, and then replated ontofibronectin coated microscope slides. The cells were cultured anadditional 24 hours to allow them to recover from passage, and thenfixed for immunocytologic analysis. Cells were reacted with MF20, amonoclonal antibody which recognizes sarcomeric myosin. Cells were thenindividually counted for the presence or absence of sarcomeric myosin, amarker for cardiomyocytes. The results were as follows:

    ______________________________________                                        Total number of cells counted:                                                                        794                                                   Number of MF20+ cells:  791                                                   Number of MF20- cells:   3*                                                   Percent cardiomyocytes: 99.6%                                                 ______________________________________                                         *The 3 cells which did not stain with MF20 may still have been                cardiomyocytes, since this antibody will not react with monomeric myosin.

It was thus demonstrated that the expression of drug (neomycin)resistance under a cardiac-specific promoter enables the selection of anessentially pure population of ES derived cardiomyocytes in culture.Such populations can be used to form myocardial grafts using proceduresas discussed in the Examples above.

EXAMPLE 5 Delivery of Protein via Graft

A. METHODS

C2C12 Cell Culture and transfection. C2C12 myoblasts (ATCC) weremaintained in the undifferentiated state by culturing at low density inhigh glucose Dulbecco's Modified Eagle Media (DMEM)¹ supplemented with20% fetal bovine serum, 1% chicken embryo extract, 100 units/mlpenicillin and 100μg/ml streptomycin. For some studies, myogenicdifferentiation was induced by culturing in DMEM supplemented with 2%horse serum and antibiotics.

A fusion gene comprised of the metallothionein (MT) promoter driving amodified Transforming Growth Factor-Beta 1 (TGF-β1) cDNA was obtainedfrom Samuel and colleagues (20). Transcriptional activity of themetallothionein promoter can be regulated by modulating the heavy metalcontent of cell culture media. The TGF-β1 cDNA carried site-directedmutations which resulted in the conversion of Cys²²³ and Cys²²⁵ toserines. This modification (described further in (21)) results ill theelaboration of a TGF-β1 molecule which is unable to form dimers, andconsequently is not subject to normal post translational regulation.Cells expressing the modified cDNA constitutively secrete processed,active TGF-β1 (20). The MT-TGF fusion gene was introduced into C2C12myoblasts by calcium phosphate transfection; stable transfectants wereselected by virtue of co-transfection with an SV40-neo^(r) transgene.Four independent clones were isolated, and presence of the transgene wasconfirmed by Southern blot analysis. The relative levels of TGF-β1expression in the different clonal cell lines was initially assessed byNorthern blot analysis, and one line, designated C2(280), was utilizedfor subsequent experiments.

Myocardial Grafting Protocol. The grafting protocol was as described inExample 1. Fourteen days, post-surgery, graft bearing animals were givenheavy metal (25 mM ZnSO₄ in drinking water). Zinc treatment wascontinued until the termination of the experiment (1-4 weeks).

Histology. For paraffin sections, hearts were fixed in 10% neutralbuffered formalin, dehydrated through graded alcohols, and infiltratedwith paraffin. Tissue blocks were then sectioned at 6μm. H and Estaining was performed directly after sectioning according tomanufacturer's specifications (Sigma Diagnostics).

For ³ H!-thymidine incorporation, mice were given a bolus and sacrificedas in Example 1. The heart was removed and processed for paraffinembedding as described above. Autoradiography was likewise conducted asin Example 1.

B. RESULTS

Expression of recombinant TGF-β1 in response to heavy metal inductionwas examined in C2(280) myoblasts and myotubes. Transgene transcripts(1.8 kb) were readily distinguished from those originating from theendogenous TGF-β1 gene (2.5 kb) by Northern blot analysis. Addition ofheavy metal to the culture media resulted in a marked increase ofrecombinant TGF-β1 transcripts in C2(280) myoblasts and myotubes. Asindicated above, modified TGF-β1 expressed by C2(280) cells should haveconstitutive biological activity. To directly test this, conditionedmedia from C2(280) myoblasts and myotubes was examined by growthinhibition assay.

C2(280) myoblasts were used to produce intra-cardiac grafts in syngeneicC3Heb/FeJ mice. The presence of grafts was readily detected in H and Estained sections. 100% (n>50) of the animals receiving intra-cardiacinjections of C2(280) cells went on to develop grafts. Interestingly, Hand E analysis suggested that the C2(280) grafts were somewhat lessdifferentiated as compared to those produced with unmodified C2C12cells. This result was confirmed by immunohistologic analysis with amonoclonal antibody which recognizes skeletal myosin heavy chain.

C2(280) graft transgene expression was assessed by immunohistology withan anti-TGF-β1 antibody; TGF-β1 expression was readily detected inC2(280) grafts. As a negative control, TGF-β1 expression was assessed ingrafts produced by C2C12 myoblasts. As expected, the relative levels ofTGF-β1 expression were markedly reduced in C2C12 grafts as compared toC2(280) grafts.

TGF-β1 is a well known angiogenic factor. ³ H-thymidine incorporationanalyses in vascular endothelial cells was therefore assessed todetermine if an enhanced angiogenic response occurred in graftsexpressing the MT-TGF transgene. DNA synthesis in vascular endothelialcells was readily apparent in C2(280) grafts under administration of asingle bolus injection of ³ H-thymidine (H-THY). In contrast, vascularendothelial DNA synthesis was markedly reduced in non-transfected C2C12grafts (Table 1). To rule out the possibility that the angiogenicresponses was due solely to graft mass, ³ H-thymidine incorporation wascompared between similar size and aged C2C12 and C2(280) grafts (Table1). A marked increase in the number of vascular endothelial cellssynthesizing DNA was apparent in all of the analyses. Finally, thethymidine incorporation assay also revealed that a percentage of thegrafted myoblasts continued to proliferate. This observation isconsistent with the known inhibitory effect of TGF-β1 onmyodifferentiation, and most likely accounts for the undifferentiatedappearance of the C2(280) grafts.

                  TABLE 1                                                         ______________________________________                                        TGF-β1 Delivery and                                                      Vascular Endothelial DNA Synthesis                                            Time Post Zn       C2Cl2      C2(280)                                         Induction          TGF-β1(-)                                                                           TGF-β1(+)                                  ______________________________________                                        1 Week   Total # .sup.3 H-THY +                                                                       0          8                                                   Endothelial Cells                                                             Total # of Vessel                                                                           35         17                                                   Sections Counted                                                              # Synthetic Cells/                                                                          0.0 ± 0.00                                                                            0.46 ± 0.036                                      Vessel Section                                                       2 Weeks  Total # .sup.3 H-THY +                                                                       1          7                                                   Endothelial Cells                                                             Total # of Vessel                                                                           18         21                                                   Sections Counted                                                              # Synthetic Cells/                                                                          0.06 ± 0.056                                                                          0.34 ± 0.052                                      Vessel Section                                                       ______________________________________                                    

While the invention has been illustrated and described in detail in theforegoing description, the same is to be considered as illustrative andnot restrictive in character, it being understood that only thepreferred embodiments have been described and that all modificationsthat come within the spirit of the invention are desired to beprotected.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

1. Tompson, L. Fetal transplants show promise. Science 257: 868-870,1992.

2. Gussoni, E., Pavlath, G. K., Lanctot, A. M., Sharma, K. R., Miller,R. G., Steinman, L. and Blau, H. M. Normal dystrophin transcriptsdetected in Duchenne muscular dystrophy patients after myoblasttransplantation. Nature 356: 435-438, 1992.

3. Steinhelper, M. E., Lanson, N., Dresdner, K., Delcarpio, J. B., Wit,A., Claycomb, W. C. and Field, L. J. Proliferation in vivo and inculture of differentiated adult atrial cardiomyocytes from transgenicmice. American Journal of Physiology 259 (Heart and CirculatoryPhysiology 28): H1826--H1834, 1990.

4. Janse, M. J., Cinca, J., Morena, H., Fiolet, J. W., Kleber, A. G.,deVries, G. P., Becker, A. E. and Durrer, D. The border zone inmyocardial ischemia. An electrophysiological, metabolic andhistochemical correlation in the pig heart. Circulation Research44:576-588, 1979.

5. Spear, J. F., Michelson, E. L., and More, E. N. Cellularelectrophysiologic characteristics of chronically infarcted myocardiumin dogs susceptible to sustained ventricular tachyarrhythmias. Journalof the American College of Cardiology 4:1099-1110, 1983.

6. Delcarpio, J. B., Lanson, N. A. Jr., Field, L. J. and Claycomb, W. C.Morphological characterization of cardiomyocytes isolated from atransplantable cardiac tumor derived from transgenic mouse atria (AT-1cells). Circulation Research 69:1591-1600, 1991.

7. Rockman, H. A., Ross, R. A., Harris, A. N., Knowlton, K. U.,Steinhelper, M. E., Field, L. J., Ross, J. Jr. and Chien, K. R.Segregation of atrial-specific and inducible expression of an ANFtransgene in an in vivo murine model of cardiac hypertrophy. Proc. Natl.Acad. Sci. USA 88:8277-8281, 1991.

8. Bullock, G. R. and Petrusz, P., in Techniques in Immunocytochemistry,Vol. II, Academic Press, New York, 1983.

9. Field, L. J. Atrial natriuretic factor-SV40 T antigen transgenesproduce tumors and cardiac arrhythmias in mice. Science 239:1029-1033,1988.

10. Katz, E., Steinhelper, M. E., Daud, A. Delcarpio, J. B., Claycomb,W. C. and Field, L. J. Ventricular cardiomyocyte proliferation intransgenic mice expressing α-Cardiac Myosin Heavy Chain-SV40 T antigenfusion genes. American Journal of Physiology 262 (Heart and CirculatoryPhysiology 31):H1867-1876, 1992.

11. Steinhelper, M. E. and Field, L. J. "SV40 large T-Antigen inducesmyocardiocyte proliferation in transgenic mice", in The development andregenerative potential of cardiac muscle, John Oberpriller and JeanOberpriller, eds. Harwood Academic press, 1990.

12. Steinhelper, M. E. and Field, L. J. Cardiac Tumors and dysrhythmiasin transgenic mice. Toxicologic Pathology 18: 464-469, 1990.

13. Yaffe, D., and O. Saxel. Serial passaging and differentiation ofmyogenic cells isolated from dystrophic mouse muscle. Nature270:725-727, 1977.

14. Nadal-Ginard, B. Commitment, fusion, and biochemical differentiationof a myogenic cell line in the absence of DNA synthesis. Cell15:885-864, 1978.

15. Bruni, C. Mitotic activity of muscle satellite cells during theearly stages of rhabdomyosarcomas induction with nickel subsulfide. InMuscle Regeneration, A. Mauro, editor. Raven Press, New York, N.Y.,1979.

16. Rubin, L. L., C. E. Keller and S. M. Schuetze. Satellite cells inisolated adult muscle fibers in tissue culture. In Muscle Regeneration,A. Mauro, editor. Raven Press, New York, N.Y., 1979.

17. B. Hogan, F. Costantini and E. Lacy, in Manipulating the MouseEmbryo--A Laboratory Manual, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1986.

18. M. E. Steinhelper, K. L. Cochrane and L. J. Field. Hypotension intransgenic mice expressing atrial natriuretic factor fusion genes.Hypertension 16:301-307, 1990.

19. A. D. Loewy, P. C. Bridgman and T. C. Mettenleiter. Brain Research555:346, 1991.

20. S. K. Samuel, et al. Autocrine induction of tumor proteaseproduction and invasion by a metallothionein-regulated TGF-beta 1(Ser223,225). Embo Journal JC:emb! 11(4):1599-1605, 1992.

21. A. M. Brunner, et al. Site-directed mutagenesis of cysteine residuesin the pro region of the transforming growth factor beta 1 precursor.Expression and characterization of mutant proteins. Journal ofBiological Chemistry 264(23):13660-13664, 1989.

22. E. J. Robertson. Embryo-derived stem cell lines, in Teratocarcinomasand embryonic stem cells: a practical approach, E. J. Robertson, editor.IRL Press, Washington D.C., 1987.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 bases                                                          (B) TYPE: Nucleotide Sequence                                                 (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGTGGGGGCTCTTCACCCCCAGACCTCTCC30                                              (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 bases                                                          (B) TYPE: Nucleotide Sequence                                                 (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (ii) MOLECULE TYPE: Genomic DNA                                               (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GCCAGGGTTTTCCCAGTCACGACGTTGT28                                                __________________________________________________________________________

What is claimed is:
 1. A method of obtaining a population of cells,comprising:transfecting embryonic stem cells to introduce a marker geneenabling selection of a cardiomyocyte cell lineage from other celllineages resulting from the differentiation of the stem cells; causingthe stem cells to differentiate in culture; and selecting saidcardiomyocyte cell lineage based on said marker gene.
 2. The method ofclaim 1, comprising:transfecting the stem cells to introduce (i) a firstmarker gene enabling selection of transfected stem cells fromnon-transfected stem cells and (ii) a second marker gene enablingselection of said one cell lineage from said other cell lineages;selecting transfected stern cells based on the first marker gene;causing the selected stem cells to differentiate; and selecting said onecell lineage based on said marker gene.
 3. An embryonic stem cell havingintroduced DNA encoding a marker gene enabling selection of acardiomyocyte cell lineage from other cell lineages resulting from thedifferentiation of the stem cells.
 4. The stem cell of claim 3 whichincludes introduced DNA encoding (i) a first marker gene enablingselection of embryonic stem cells having the first marker gene fromembryonic stem cells not having the first marker gene and (ii) a secondmarker gene enabling selection of said cardiomyocyte cell lineage fromsaid other cell lineages.
 5. The stem cell of claim 3, wherein saidmarker gene confers antibiotic resistance to cells which express themarker gene.
 6. The stem cell of claim 4, wherein said first and secondmarker genes each confer antibiotic resistance to cells which expressthe marker genes.
 7. The stem cell of claim 6, wherein said secondmarker gene is under the control of a promoter which causes expressionof the marker gene only in said cardiomyocyte cell lineage.
 8. A methodof obtaining a population of cells, comprising:providing an embryonicstem cell having introduced DNA encoding a marker gene enablingselection of a cardiomyocyte cell lineage from other cell lineagesresulting from the differentiation of the stem cells; causing the stemcell to differentiate in culture to form a mixed differentiated cellularpopulation containing said a cardiomyocyte cell lineage and said othercell lineages; selecting from the cellular population said acardiomyocyte cell lineage from said other cell lineages based on saidmarker gene.
 9. The method of claim 8 in which:said marker gene is underthe control of a promoter which causes expression of the marker gene insaid cardiomyocyte cell lineage but not in said other cell lineages, soas to enable selection of said cardiomyocyte cell lineage from saidother cell lineages.
 10. The method of claim 9, in which said markergene confers antibiotic resistance, and wherein said selecting includescontacting the mixed differentiated cellular population with anantibiotic to which cells of said one cell lineage have been conferredresistance by the expression of said first marker gene, thereby killingcells of said other cell lineages but not cells of said one cell lineagewhich express the marker gene.
 11. The method of claim 9, in which saidmarker gene confers resistance to neomycin or hygromycin.
 12. Apopulation of cardiomyocyte cells obtained by a method according toclaim
 8. 13. The population of cells of claim 12, which is obtained by amethod according to claim 10.